The present invention relates to an assay for the detection of sodium channel-specific toxins, particularly marine toxins, based upon mitochondrial dehydrogenase activity in the presence of ouabain and veratridine. More specifically, the present invention concerns a cell bioassay that allows the detection of either sodium channel blocking agents, such as saxitoxin, or sodium channel enhancers like brevetoxin and ciguatoxin.
Commercially important species of shellfish and finfish are known to occasionally present a serious health risk to consumers due to the presence of accumulated marine toxins. A significant number of these marine toxins exert their effects by interaction with voltage sensitive sodium channels in excitable membranes. Paralytic shellfish poisoning (PSP) is attributed to the ingestion of molluscan shellfish that have accumulated saxitoxins or related compounds from toxic dinoflagellate blooms. The saxitoxins selectively block ion transport at the sodium channel. Neurotoxic shellfish poisoning (NSP) is caused by the ingestion of shellfish that have sequestered brevetoxins from the dinoflagellate associated with Florida's red tide. In contrast to the action of saxitoxin, brevetoxins perturb normal membrane properties of excitable cells by activating sodium channels. Ciguatoxins, which accumulate in tropical fish, also exert their biological effects through activation of sodium channels.
Monitoring programs for marine toxins have depended in large part upon mouse bioassays. Although mouse bioassays have for many years provided a fairly reliable assessment of risk, there is mounting pressure to develop alternative assays to reduce the reliance on animal testing. To this end Kogure et al. developed a tissue culture assay for tetrodotoxin, saxitoxin, and related toxins. See Kogure et al., Toxicon 26 (2): 191-97 (1988). In the Kogure assay, a mouse neuroblastoma cell line (Neuro-2a) is treated with a fixed concentration of the sodium channel activator veratridine in the presence of ouabain, an inhibitor of Na+/K+ ATPase. The combined effect of these agents is an enhanced sodium influx, leading to altered cell morphology, a subsequent decrease in cell viability and ultimate cell lysis. Tetrodotoxin, saxitoxin and related toxins which block sodium channels antagonize this effect, essentially rescuing the cells in a dose dependent manner. This phenomenon provides the basis of a sensitive in vitro bioassay for these toxins. But evaluation of the Kogure assay requires the visual scoring of 200 or more cells per sample or well, a potentially time-consuming and operator dependent task.
Scoring of this assay was improved by the modifications described by Jellett et al., Toxicon 30 (10): 1143-56 (1992), the contents of which are hereby incorporated by reference. Jellett et al. used a microplate reader for automated determinations of absorbances of toxin-treated cells which were stained with crystal violet. This assay exploits the difference in adherence to the culture well of cells treated only with ouabain/veratridine and PSP toxin-treated cells. The former cells exhibit diminished adherence to the culture well, associated with swelling and lysis, and thus are readily removed by rinsing, whereas the latter retain substrate adherence. Thus, cells affected only by ouabain/veratridine lose adherence and are removed during rinsing, while cells inoculated with the toxin, and thus protected from the effects of ouabain/veratridine, remain in the well.
In the Jellett assay, wells containing Neuro-2a cells are inoculated with toxin and then with ouabain/veratridine, incubated, and subsequently rinsed. After rinsing, the wells are fixed and subsequently stained with crystal violet. The processed plates are then dried, followed by digestion of the stained cells in acetic acid. Finally, the plates are read for absorbance of crystal violet in each well, with the absorbance being directly related to the amount of PSP toxin originally present. These modifications notably improve the application of the cell bioassay developed by Kogure et al. But the numerous steps involving mechanical removal of cells and treatment of the plates may be subject to operator variability.
Thus, a simplified bioassay for detecting the above-described toxins would be very useful. It also would be desirable to have a bioassay for detecting other types of toxins, especially those marine toxins that are sodium channel activators like the brevetoxins and the ciguatoxins. Since the known tissue culture-based bioassays are limited to detection of sodium channel blocking agents, there is a real need for a tissue culture-based bioassay which can detect marine toxins which have sodium channel activating properties as well display sodium channel blocking activity.